RNA-Based CTC Analysis Provides Prognostic Information in Metastatic Breast Cancer

Abstract

In metastatic breast cancer (MBC) the molecular characterization of circulating tumor cells (CTCs) provides a unique tool to understand metastasis-biology and therapy-resistance. We evaluated the prognostic significance of gene expression in EpCAM(+) CTCs in 46 MBC patients based on a long follow-up. We selected a panel consisting of stem cell markers (CD24, CD44, ALDH1), the mesenchymal marker TWIST1, receptors (ESR1, PGR, HER2, EGFR) and the epithelial marker CK-19. Singleplex RT-qPCR was used for TWIST1 and CK-19 and multiplex RT-qPCR for stem cell markers and receptors. A group of 19 healthy donors (HD) was used as control. Univariate (p = 0.001) and multivariate analysis (p = 0.002) revealed the prognostic value of combined gene expression of CK-19(+), CD44high/CD24low, ALDH1high/CD24low and HER2 over-expression for overall survival (OS). The Kaplan–Meier estimates of OS were significantly different in patients positive for CK-19 (p = 0.028), CD44high/CD24low (p = 0.002), ALDH1high/CD24low (p = 0.007) and HER2-positive (p = 0.022). Our results indicate that combined gene expression analysis in EpCAM(+) CTCs provides prognostic information in MBC.

Comparison of microfluidic platforms for the enrichment of circulating tumor cells in breast cancer patients

“…In addition to gene expression analysis using Taqman®-based qPCR following target-specific pre-amplification, a highly specific assay for the detection of CK19 transcripts in CTCs using the hybridization probe-based Lightcycler technology was employed [ 16 ]. In line with Strati et al and using their set of primers and probes in metastatic BC [ 17 ], 21.1% CK-positive cases in the same setting were observed. Moreover, the results from the Lightcycler qPCR reasonably agree with the Taqman® hydrolysis probe-based common qPCR in detecting CK19 transcripts (Cohen’s κ = 0.74), alleviating concerns regarding a potential bias and lower specificity introduced by the target-specific pre-amplification step preceding Taqman® qPCR.…”

Detection and Molecular Characterization of Circulating Tumour Cells: Challenges for the Clinical Setting

“…TLR4 and pSTAT3 are key players in cancer inflammation and immune evasion; in MBC TLR4-pos CTCs correlated with a high risk of disease progression [ 46 ]. In MBC, RT-qPCR analysis in CTC-derived RNA for stem-cell markers ( CD24 , CD44 , ALDH1 ), TWIST1 , ESR1 , PGR , HER2 , EGFR and CK-19 revealed that the combined gene expression of CK-19 (+), CD44 high /CD24 low , ALDH1 high /CD24 low and HER2 overexpression was correlated with OS [ 47 ]. A 17-gene digital RNA signature of breast CTC-derived transcripts was reported to enable early monitoring of response with high sensitivity.…”

Signatures of Breast Cancer Progression in the Blood: What Could Be Learned from Circulating Tumor Cell Transcriptomes

“…Two 8-gene signatures able to predict response to therapy, and overall including CXCL14 , KRT7 , KRT19 , KRT81 , PKP3 , PTRF , TIMP3 , LAD1 , S100A16 , FKBP10 , TWIST1 , PTRF , EEF1A2 , PTPRK , EGFR and ERBB3 genes, were generated by comparing the CTC profile of poor and good responders following first line treatment with standard hormonal or chemotherapy or with aromatase inhibitors only [ 166 , 167 ]. Other CTC-detected genes involved in drug resistance, such as MTOR , PIK3CA , ADAM17 , ALDH2 , TIMP1 , PALB2 and MYC , in addition to overexpressed epithelial and EMT-related markers, such as EpCAM high , CD44 high , and ALDH1 high , may have prognostic significance or can be found more frequently expressed in poor responders [ 136 , 137 , 168 , 169 , 170 ].…”